The NINDS Anticonvulsant Screening Program (ASP) has identified most of the anticonvulsants in clinical use today. However, one third of epileptic patients do not respond to these drugs. The ASP protocols are based on seizures induced by subjecting normal animals to acute convulsant conditions. We have developed a complimentary system of novel in vitro and in vivo assays of spontaneous seizures in chronically epileptic preparations. This two-stage screening system provides a unique focus on recurrent spontaneous seizures in chronic epilepsy models. The first stage is an in vitro assay comprised of the organotypic hippocampal slice culture, which develops electrographic seizure activity and corresponding biochemical biomarkers over the first week in vitro. The second stage is an in vivo assay comprised of the kainate model of epilepsy in which spontaneous seizures are monitored using continuous telemetry and supervised, blinded, computerized seizure detection. We used the rapid in vitro assay to screen over 400 compound-concentration combinations from the NINDS Custom Compound Collection. We found a lead compound, celecoxib, and then verified this lead by the second-stage testing in a randomized double blind in vivo crossover trial. Celecoxib had no effect on seizures induced by acute application of convulsants to normal brain tissue, suggesting that its anticonvulsant properties are unique to chronic epilepsy, and raising the possibility that its spectrum of action will be distinct from anticonvulsants discovered by the ASP protocols. The next step in development is medicinal chemistry to optimize celecoxib?s anticonvulsant efficacy. This is most feasibly accomplished through the UH2 / UH3 Blueprint Neurotherapeutics Network. As our discussions with BPN program officers clarified, to efficiently utilize the BPN medicinal chemistry program we must further develop the in vitro and in vivo assays and acquire additional data on our lead compound. The UH2/3 mechanism was considered the most appropriate funding mechanism by the NINDS program officer. In the R21 phase of this proposal, we will extend the in vitro assay?s concentration-response for celecoxib and 2,5 dimethyl celecoxib, a derivative that does not inhibit COX2 but has equal anticonvulsant efficacy in vitro. We will then characterize the assay?s reproducibility and Z factor. We will also establish the dose-response of the in vivo assays for celecoxib, and increase the number of in vitro and in vivo sites to two each in order to improve robustness and throughput, as well as engage outstanding younger investigators in this effort. In the R33 phase of the proposal, we will further characterize the lead compound by determining whether COX2 inhibition is necessary for anticonvulsant activity in the in vitro and in vivo assays.
Anticonvulsant screening technologies based on acute application of convulsant conditions to normal animals do not capture the full spectrum of conditions that lead to seizures in chronic epilepsy, and as a consequence one third of epileptic patients remain medically intractable. We have used novel assays based on chronic epilepsy to screen drugs for anticonvulsant activity and have identified 2 lead compounds, celecoxib and a derivative that does not have celecoxib?s systemic effects. Here we propose to optimize our in vitro and in vivo assays for the NIH Blueprint Therapeutics medicinal chemistry program, with the goal of bringing a more specific derivative of celecoxib to clinical trials.
